In many biomedical fields it is required to record acute signaling events which reflect biological pathways or mechanisms. One common method of monitoring gene expression in a living cell, tissue or animal relies on the use of a luminescent reporter gene placed under the control of a gene of interest in said cell, tissue or animal. The activity of genes can be visualized in real-time either by fluorescence or bioluminescence recording of transgenic cells or organisms expressing fluorescent proteins or luciferase, respectively.
Both of these techniques have been successfully adapted to whole-body imaging of mice (Hoffman and Yang (2006) Nat. Protoc 1, 1429-1438; Sandhu et al. (2010) Wiley Interdiscip Rev Syst Biol Med 2, 398-421). However, in most applications the animals were anesthetized to allow for long exposure times and expressed reporter genes that were driven by strong, constitutively active promoters and that specified mRNAs and/or proteins with long half-lives. As the steady-state accumulation of an mRNA or a protein equals synthesis rate (S) times half-life (T1/2) divided by the natural logarithm of 2 (ST1/2/In2), and since the number of photons is directly proportional to the cellular concentration of the fluorescent or luminescent proteins, such reporter genes produce strong and thus readily detectable signals.
For particular studies, such as the recording of circadian gene expression, the genes are less potently transcribed and the reporter gene products (mRNA and protein) are short-lived. As a result, the luminescent or fluorescent proteins accumulate to relatively low concentrations and hence produce only relatively weak signals. Yet frequent image acquisition over extended time periods (several days) are required in such studies, and this obviously precludes anesthesia (and thus long exposure times) of the animals under investigation. Moreover, conventional fluorescence measurements imply excitation with bright light, which would be likely to phase-shift animals during the recording. Therefore, bioluminescence monitoring would be a more judicious method for tracking rhythmic gene expression.
The IVIS Kinetic® system from Caliper Life Sciences provides a real-time, fast imaging system enabling acquisition of biologically relevant events within milliseconds. However, the signal can only be detected for a few minutes at most. When combined with an isolation chamber, this system allows bioluminescence to be monitored in conscious animals.
Despite its usefulness, the technology of the prior art cannot be applied to experimental conditions where bioluminescence is expressed in tissues containing a small number of cells, such as skin, skeletal and heart muscle, kidney, parts of the intestine, spleen, and exocrine pancreas, nor does it allow, for instance, the long-term monitoring of circadian gene expression in tissues of freely moving individual animals.
With the technology of the prior art, signal strength may be a limiting factor as well as the duration of the monitoring of the bioluminescence signal. Moreover, the technology of the prior art does not allow the effect of controlled environmental conditions including food, light, medicaments, etc. to be studied.
Therefore, there is still a need to further provide a technology and apparatus for real-time in vivo bioluminescence monitoring in a small animal allowing a high temporal resolution, with a minimal background noise, on a long term such as several days or several weeks, and in controlled environmental conditions.
The apparatus and method of the invention solve this problem and could find applications in circadian, ultradian, or infradian biology, for instance, but should also, without being limited to these examples, readily reveal the kinetics of signaling by hormones, cytokines, neuronal pathways, and metabolites such as bile acids, cholesterol, fatty acids, glucose, oxygen, and medical drugs, or allow measurement of the expression of the genes of the cellular cycle in sane and cancerous tissues, or the expression of the genes related to physical activity.